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Recombinant prespore-specific antigen from Dictyostelium discoideum is a beta-sheet glycoprotein with a spacer
N E Zachara1, N H Packer, M D Temple
1Macquarie University Centre for Analytical Biotechnology, Sydney, Australia.
Abstract:
Prespore-specific antigen (PsA) is a putative cell-adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell-surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (glycopeptide) that is attached to the cell membrane through a glycosyl-PtdIns anchor. The sequence of PsA suggests that PsA may belong to a new family of cell-surface molecules and here we present information on the structure of the N-terminal globular domain and determine the reducing-terminal linkage of the O-glycosylation. To obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D. discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of beta-sheet in the N-terminal globular domain, a feature commonly observed in cell-adhesion proteins. Solid-phase Edman degradation of the glycopeptide of rPsA indicated that 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structures of the O-glycosylations were determined to be single N-acetylglucosamine residues.
Insights
Prespore-specific antigen (PsA) in Dictyostelium discoideum features a beta-sheet rich N-terminal domain. Its O-glycosylated stem consists of single N-acetylglucosamine residues, revealing insights into cell-adhesion molecule structures.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Prespore-specific antigen (PsA) is a cell-adhesion molecule in Dictyostelium discoideum.
- PsA shares structural similarities with mammalian cell-surface proteins, suggesting a conserved functional role.
- Its structure includes an N-terminal globular domain, an O-glycosylated stem, and a glycosyl-PtdIns anchor.
Purpose of the Study:
- To elucidate the structure of the N-terminal globular domain of PsA.
- To determine the reducing-terminal linkage of O-glycosylation in PsA.
- To characterize a secreted recombinant form of PsA (rPsA) for detailed analysis.
Main Methods:
- Expression and characterization of recombinant PsA (rPsA) in Dictyostelium discoideum.
- 1H-NMR spectroscopy to analyze the secondary structure of the N-terminal domain.
- Solid-phase Edman degradation to identify glycosylated residues in the glycopeptide.
Main Results:
- 1H-NMR spectra indicated a high degree of beta-sheet structure in the N-terminal globular domain of rPsA.
- Solid-phase Edman degradation revealed that 14 out of 15 threonines and serines in the spacer region were glycosylated.
- The O-glycosylations were identified as single N-acetylglucosamine residues.
Conclusions:
- The N-terminal domain of PsA possesses a beta-sheet rich structure, common in cell-adhesion proteins.
- PsA is extensively O-glycosylated with single N-acetylglucosamine residues on its stem.
- These findings contribute to understanding the structure and potential function of this novel cell-surface molecule and related proteins.